In FY16, we have continued to address the role of TGF-beta in regulating the cancer stem cell (CSC) compartment using a novel functional imaging approach that we developed to allow visualization of this minority cell population in real time and in situ. Our lentiviral-based CSC reporter uses a synthetic promoter in which expression of a fluorescent protein is driven by the stem cell master transcription factors Oct4 and Sox2. Using this approach, we have developed methods that allow extended cell fate mapping of individual CSCs in 3D culture systems and in lung slice models ex vivo. We are now addressing the effect of TGF-beta superfamily members on CSC proliferation, survival and motility in breast cancer model systems that show either tumor suppressor or pro-progression responses to TGF-beta. Using a xenograft breast cancer model in which TGF-beta functions as a tumor suppressor, we have identified a subpopulation of CSCs with high endogenous TGF-beta signaling that were intrinsically less proliferative than the other CSCs, suggesting that TGF-beta may be important in maintaining a fraction of CSCs in the quiescent state. Exogenous TGF-beta selectively inhibited asymmetric self-renewing mitoses in the CSC compartment, and specifically inhibited the invasion of CSCs through basement membrane. Consistent with these observations, in vivo treatment with TGF-beta neutralizing antibodies increased the CSC content of two xenograft models of well-differentiated breast cancer. Parallel work is ongoing in models of more aggressive advanced disease. We have a major collaboration with the Condeelis Lab at AECOM to use intravital videomicroscopy to observe the localization and behavior of CSCs in real time in the primary tumor and metastatic lungs of mouse xenograft or syngeneic allograft breast cancer models. Understanding how CSCs are regulated in vivo will be critical to development of more effective cancer therapies, as these cells are largely resistant to existing therapeutic approaches. We are employing integrated genomic approaches including RNA-Seq and ATAC-Seq to address the molecular mechanisms underlying differential effects of TGF-beta on CSC and non-CSC compartments at early and late stages of the disease process. Finally we are also using our cancer stem cell reporter to develop a medium-to-high throughput drug screen to test the NCI Natural Product Repository for novel agents that selectively inhibit the cancer stem cell.